Purification and Characterization of Agarase from Marine Bacteria Acinetobacter sp. PS12B and Its Use for Preparing Bioactive Hydrolysate from Agarophyte Red Seaweed Gracilaria verrucosa.

Acinetobacter strain PS12B was isolated from marine sediment and was found
to be a good candidate to degrade agar and produce agarase enzyme. The extracellular
agarase enzyme from strain PS12B was purified by ammonium sulfate precipitation
followed by DEAE-cellulose ion-exchange chromatography. The specific activity of the
crude enzyme which was 1.52 U increased to 45.76 U, after two-stage purification, with
an enzyme yield of 9.76%. Purified enzyme had a molecular mass of 24 kDa. The
optimum pH and temperature for activity of purified agarase were found to be 8.0 and
40 °C, respectively. The Km and Vmax values for agarase were 4.69 mg/ml and
0.5 μmol/min, respectively. Treatment with EDTA reduced the agarase activity by
58% at 5 mM concentration. The enzyme activity was stimulated by the presence of
Fe2+, Mn2+, and Ca2+
ions while reducing reagents (β-mercaptoethanol and
dithiothreitol, DTT) enhanced its activity by 30–40%. The purified agarase exhibited
tolerance to both detergents and organic solvents. Major hydrolysis products of agar
were DP4 and also a mixture of longer oligosaccharides DP6 and DP7. The enzyme
hydrolysed seaweed (Gracilaria verrucosa) exhibited strong antioxidant activity
in vitro. Successful hydrolysis of seaweed indicates the potential use of the enzyme to
produce seaweed hydrolysate having health benefits as well as the industrial application
like the production of biofuels.